Quantifying the relationship between temperature regulation in the ear and floret development stage in wheat (Triticum aestivum L.) under heat and drought stress

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Steinmeyer, F. T., Lukac, M., Reynolds, M. P. and Jones, H. E. (2013) Quantifying the relationship between temperature regulation in the ear and floret development stage in wheat (Triticum aestivum L.) under heat and drought stress. Functional Plant Biology, 40 (7). pp. 700-707. ISSN 1445-4408 doi: https://doi.org/10.1071/FP12362 Available at http://centaur.reading.ac.uk/32198/

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Quantifying the relationship between temperature regulation in the ear and floret 1

development stage in wheat (Triticum aestivum L.) under heat and drought stress 2

3

Authors: F.T. Steinmeyer1, M. Lukac

1, M.P. Reynolds

2, H.E. Jones

1* 4

5

1 School of Agriculture, Development and Policy, University of Reading, Reading RG6 6AR, 6

UK. 7

2 International Maize and Wheat Improvement Centre (CIMMYT), Int., AP 6-641, 06600 8

Mexico, DF, Mexico. 9

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Corresponding author (*): H.E. Jones (h.e.jones@reading.ac.uk) 11

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Keywords: Wheat, anthesis, temperature depression, controlled environment, screening 14

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Summary Text for Table of Contents: 16

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The relationship between temperature depression in the ears of Triticum aestivum L. and 18

flower development stage under heat and drought stress was examined. The early stages of 19

anthesis were associated with a lower ear temperature than the latter stages, indicating that 20

temperature depression occurs in the ear under stressed conditions, and potentially during the 21

heat sensitive flower development stages. This pioneering study provides a framework for 22

correlating ear temperature and grain yield under stressed conditions. 23

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Abstract 35

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Thermal imaging is a valuable tool for the elucidation of gas exchange dynamics between a 37

plant and its environment. The presence of stomata in wheat glumes and awns offers an 38

opportunity to assess photosynthetic activity of ears up to and during flowering. The 39

knowledge of spatial and temporal thermodynamics of the wheat ear may provide insight into 40

interactions between floret developmental stage (FDS), temperature depression (TD) and 41

ambient environment, with potential to be used as a high-throughput screening tool for 42

breeders. A controlled environment study was conducted using six spring wheat (Triticum 43

aestivum L.) genotypes of the elite recombinant inbred line Seri/Babax. Average ear 44

temperature (AET) was recorded using a hand held infrared camera and gas exchange was 45

measured by enclosing ears in a custom built cuvette. FDS was monitored and recorded daily 46

throughout the study. Plants were grown in pots and exposed to a combination of two 47

temperature and two water regimes. In the examined wheat lines, TD varied from 0.1°C to 48

0.6°C according to the level of stress imposed. The results indicated that TD does not occur 49

at FDS F3, the peak of active flowering, but during the preceding stages prior to pollen 50

release and stigma maturity (F1-F2). These findings suggest that ear temperature during the 51

early stages of anthesis, prior to pollen release and full extension of the stigma, are likely to 52

be the most relevant for identifying heat stress tolerant genotypes. 53

54

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3

Introduction 56

57

On-going alteration of the global climate is predicted to lead to an increase in the frequency 58

of extreme weather events such as heat waves and droughts (IPCC 2007). The challenge 59

facing crop breeders is to create food crops with increasing resilience to environmental stress, 60

whilst producing ever higher yields. The full exploitation of the crop’s genetic potential is 61

vital to achieve optimal crop performance. However, the ability of a plant to yield under 62

extreme or variable environmental conditions is actually mediated by a more complex 63

phenotype. Multiple points in the plant’s development may exhibit various forms of 64

resilience, including early flowering (Acevedo et al. 2002), deep rooting (Hurd 1968), waxy 65

leaves (Cameron et al. 2006), as well as pollen production (Bita et al. 2011). 66

67

In wheat, anthesis is thought to be especially vulnerable to environmental stress (Saini and 68

Aspinall 1982). There are two approaches that wheat breeders can utilise to increase the 69

resilience of a wheat crop to environmental stress during anthesis: avoidance/escape or 70

tolerance. Lukac et al. (2012) concluded that by extending the period of flowering in wheat, 71

plants may be able to mitigate the effect of adverse environmental conditions at flowering by 72

staggering floret development. A plant can reduce the risk of a high temperature incident 73

(above 28°C) occurring and damaging all florets simultaneously by extending the flowering 74

period. Losses can be limited by having only a few florets at sensitive stages of development 75

at any one time. Adapting the flowering phenology to cope with environmental stress utilises 76

the avoidance/escape mechanism. Alternatively, tolerance allows a plant to develop in 77

conditions of environmental stress through mechanisms that actively shield key processes 78

from abiotic stresses (Wahid et al. 2007). Prior to anthesis, the process most sensitive to 79

environmental stress is the development of the male reproductive gamete. Pollen formation is 80

severely impaired by temperatures above 30°C for as little at 72 hours (Saini et al. 1983). 81

Heat tolerant lines of wheat have been shown to possess lower canopy temperature (CT) than 82

susceptible lines, achieved by a higher rate of transpiration in the canopy (Pinto et al. 2010). 83

The cooling of the canopy may be an active process and has evolved to shield the plants from 84

extreme temperatures during the most sensitive stages of development, or be merely a passive 85

indicator of improved physiology under stress environments. An increase in evaporative 86

cooling by the rest of the plant, however, will not have a direct effect on the temperature of 87

florets. 88

89

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Under drought stress, the photosynthetic activity within the awns has been found to make a 90

significantly greater contribution to the total assimilate within the ear (Evans et al. 1972). The 91

same study demonstrated that the contribution of the awns to total grain yield only occurred 92

when plants were grown under stress. Photosynthesis in the glumes and awns of a wheat plant 93

can provide up to 30% of total grain carbon under ambient conditions and it has been 94

suggested that increases in ear photosynthesis will result in increased yield (Parry et al. 95

2011). The physiological effects of heat and drought stress on canopy leaves are numerous 96

and have been well documented (Al-Katib and Paulsen 1984; Berry and Bjorkman 1980; 97

Blum 1986). However, in the view of the potential contribution of spikes to the overall gas 98

exchange of the plant and a direct link to heat sensitive processes during anthesis, spike 99

temperature dynamics may offer a great potential in identifying phenotypes specific to stress 100

tolerance at anthesis. Significant gaps exist in our knowledge of the interaction between the 101

floret temperature and ambient environment. 102

103

Flowering in wheat is not a uniform process that occurs at an even rate along the ear. In order 104

to conserve resources, temperature depression (TD) may only occur in different sections of 105

the ear when the critical stages of stigma and anther development are occurring. Lukac et al. 106

(2012) identified significant differences in the pattern and rate of floret development within 107

and between spikes on the same plant. If TD takes place in the ear, one explanation for its 108

temporal variation may be the total number of florets at a critical floret development stage 109

(FDS) in the ear. This suggestion is supported by findings by Karimizadeh and Mohammadi 110

(2011), who concluded that canopy temperature depression (CTD) takes place at varying 111

rates depending on the growth stage of the plant. Although the vast majority of studies 112

investigating photosynthetic rate and environmental stress have been conducted on plant 113

canopy, their conclusions should be applicable to the photosynthetic tissue of the ear. Ear 114

temperature depression (ETD) denotes the difference between the air temperature and ear 115

temperature and may be expressed by the following formula; 116

117

ETD = Ta – Te 118

119

where Ta is the air temperature and Te is the ear temperature. ETD will have a positive value 120

when the ear temperature is lower than that of the air. ETD is a physiological trait potentially 121

useful to breeders aiming to screen genotypes for their ability to protect crucial stages of 122

development from environmental stress. 123

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Lawlor (2009) postulated that impacts of environmental stress on plants result in a number of 124

short and long-term responses, all with the ultimate goal of acclimatising the plant and 125

ensuring its survival. The effect of heat and drought stress on the physiological and metabolic 126

activities in plants has been studied in great detail in recent years (Chaves et al. 2009; Lu and 127

Zang 2000; Mittler 2006; Wang et al. 2003). Many controlled environment studies have 128

focused on the effects of a single abiotic stress factor on the plant (Mittler 2006). Under field 129

conditions, however, multiple stress factors affecting plant development and photosynthesis 130

are compounded. The occurrence of abiotic stress is difficult to forecast more than a few 131

weeks in advance and may occur both early and late in the season. Breeders currently use a 132

range of screening methods, such as root morphology, CT, photosynthetic activity and days 133

until maturity, to select for stress tolerance (Reynolds 2002). A screening tool for stress 134

tolerance based on floret and/or ear temperature regulation does not currently exist, but may 135

be relevant when breeding plants for stress tolerance during anthesis. Before such a 136

potentially effective high-throughput screening tool for breeders is developed, it is crucial to 137

quantify the strength of interactions between the ear and the ambient environment. In order to 138

assess the scope of using ETD as a screening tool, this study sets out to detail the interaction 139

between floret development stage (FDS) and environmental stress and to study the 140

mechanisms of temperature depression (TD) utilized by the ear in stressed conditions. Four 141

key hypotheses were tested in this study; (1) genotypes tolerant to abiotic stress will have a 142

lower AET and therefore minimise damage to florets during anthesis resulting in higher grain 143

yields; (2) the basal section of the ear will be cooler than the middle section, which in turn is 144

cooler than the apical section due to its proximity to the terminal node on the stem; (3) stress 145

tolerant lines will increase the photosynthetic rate of the ear when the florets are at FDS F3; 146

and (4) in stress tolerant lines, the expected increase in photosynthetic activity of the ear at 147

FDS F3 will minimise damage to the plants reproductive organs resulting in higher grain 148

yields. 149

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Materials and methods 158

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Plant material and controlled environment description 160

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Six recombinant inbred lines (RIL) of Mexican spring wheat were studied in controlled 162

environment (CE) conditions at the Plant Environment Laboratory, University of Reading 163

(UK). The plant material originated from a reciprocal crossing of two related parent lines, 164

namely ‘Seri M82’ (IWIS CODE (Fox et al. 1996), selection history: M31 IBWSN S-1 165

MXI96-97) and ‘Babax’ (IWIS CODE (Fox et al. 1996), selection history: CM92066-J-0Y-166

0M-0Y-4M-0Y-0MEX-48BBB-0Y). Both are considered to be highly adapted semi-dwarf 167

lines (CIMMYT Wheat Personnel 1986), with Babax being highly tolerant to severe drought 168

whereas Seri M82 is moderately susceptible to severe drought (Pfeiffer 1988). Known as 169

Seri/Babax, this cross is widely used for phenotyping studies in heat and drought stress 170

environments. Seri/Babax has a relatively short period of flowering between 10 and 15 days, 171

making it ideal for this type of work (Olivares-Villegas et al. 2007). The lines used in this 172

study were Seri/Babax SB009, SB020, SB087, SB118, SB155 and SB165. Based on their 173

contrasting performance in field conditions under heat stress, as well as their similar 174

phenology and field performance without stress, Pinto et al. (2010) suggested pairing the 175

following contrasting lines of Seri/Babax: SB009/SB118, SB020/SB087 and SB155/SB165. 176

As this was a pioneer study, such pairs with contrasting phenology and performance were 177

utilised due to the fact that any observed differences are likely to be more informative of the 178

studied mechanism than when comparing lines with different phenologies. 179

180

Three seeds from each of the six lines were sown into 180mm plastic pots containing a 181

sterilised mixture of vermiculite, sand, gravel and compost (2: 1: 2: 0.5 ratio) as well as 2 182

kg/m³ Osmocote slow release granules containing N:P205:K2O:MgO (15: 11: 13: 2 ratio). 183

Half of the pots were irrigated to field capacity (FC) three times daily (Irr) by an automated 184

drip system. The other half received minimal water to simulate drought conditions (Dro), 185

which was defined as ‘infrequent irrigation such that the water applied to the pot resulted in 186

the potting mix reaching no more than 25% of the FC at any given time’. The drought 187

treatment averaged 75 ml of irrigation every two days. Soil moisture content was monitored 188

by rotating twelve automatically logged theta probes (Delta-T Devices, Cambridge, UK) 189

between pots in all four cabinets on a daily basis. In the drought treatment, soil was 190

considered sufficiently dry when the voltmeter readings were between 100 and 120mV (18.7-191

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22.4% of FC). The soil in the Irr treatments was considered wet when the soil had a voltmeter 192

reading of between 275 and 500mV (51.4-93.5% of FC), with field capacity (FC) being 193

identified as being at 535mV. The plants were irrigated with an acidified complete nutrient 194

solution, containing 100mgL-1

inorganic nitrogen. Although the potting mix selected for this 195

experiments means that results may be difficult to translate to field conditions, the intention 196

was to ensure free drainage of water from the pots in the growth cabinets so that drought 197

conditions can be easily simulated. A drying out curve of the potting mix in controlled 198

environment conditions was plotted (Supplementary Figure S1) under constant abiotic 199

conditions of 20°C. The water retention capacity of the potting mix mediated a ca. 25% 200

decline from FC over the initial 24 h period, whilst over 48 h the pots lost 32% of FC. 201

Electrical conductivity (EC) was followed during the drying process and remained within the 202

acceptable range for suitably wet soil over a 24 h period and did not reach values indicative 203

of water stress. Given that pots were irrigated to FC every 24 hours, the observed pattern of 204

water loss indicates that sufficient water remained accessible to plants between irrigation 205

events in the Irr treatment. 206

207

The plants were grown outdoors under bird netting until GS39 (Zadoks et al. 1974), when the 208

growth of plants in each pot was restricted to two plants per pot and two tillers per plant. 209

Once 50% of tillers had reached GS58-59, the pots were randomly allocated to four 1.37 x 210

1.47 m2 Saxcil growth cabinets. Two cabinets were maintained at 28°C/18°C day/night cycle 211

(‘Hot’ treatment) and the other two were maintained at 22°C/14°C day/night cycle (‘Cool’ 212

treatment), with a margin of error of ±0.5ºC. The photoperiod lasted for 16h at 650µmol m-² 213

s-1

. The plants were kept in the growth cabinets until flowering was complete (Zadoks 214

Growth Stage 69) and senescence had begun (Zadoks Growth Stage 70). 215

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Flowering and ear physiology measurement 217

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Due to flowering synchrony between the sides of the ear (Lukac et al. 2012) only the florets 219

on the even side were scored to determine the floret development stage (FDS). The 220

developmental stages of individual florets were scored after Lukac et al. (2012), with four 221

stages of anther development (Supplementary Fig. S2) and three stages of stigma 222

(Supplementary Fig. S3) identified in each floret at each sampling date. The method allows 223

for a quick identification of the stages of floral development for both the stigma and anther. 224

Pollination occurs when the stigma is at stage F and the anther is at stage 3 (FDS F3). The 225

8

odd side of the ear was not scored during any stage of the growth cycle and was reserved for 226

infrared (IR) imaging. This was done to prevent damage to the glumes and interference with 227

the temperature readings of the florets. IR images were taken daily using a hand held, thermal 228

imaging camera (FLIR Systems, Oregon, USA) between 09.00h and 12.00h for a total of six 229

days (until the end of anthesis). The IR camera used (FLIR model T335) operated in a 230

spectral range of 7.5 to 13 µm and was accurate to ±2% of the reading (FLIR 2013). In order 231

to avoid any interference with the temperature of the ear, the pot was turned within the 232

cabinet so that the odd side faced the camera whilst ensuring that the ear was not touched. 233

The camera was held horizontally between 30 and 35cm away from the ear in the growth 234

cabinet when the reading was taken. Thermal image background did not interfere with the ear 235

temperature readings. 236

237

Gas exchange measurements at ear level were conducted using CIRAS 1 (PP Systems, 238

Ayrshire, UK), a portable gas exchange analyser. Net carbon dioxide flux and relative 239

humidity were recorded in a specially constructed, clear and sealed cuvette placed around the 240

ear during analysis. Measurements of CO2 concentration and relative humidity inside the 241

cuvette took place at 10s intervals, for a total of 100s (10 readings in total). In each growth 242

cabinet,four second order tillers per line were randomly selected and followed throughout the 243

photosynthesis recordings. Only ears that had not been scored were used for gas exchange 244

measurement. Recordings were taken for three consecutive days during morning (09.00h-245

11.00h), midday (12.00h-14.00h) and afternoon (15.00h-17.00h). This terminology was 246

chosen to denote distinct periods within the diurnal cycle. The plants in controlled 247

environment cabinets experienced a significant temperature and light gradient during the 248

day/night transitions, analogous to ambient conditions. 249

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Statistical data analysis 251

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Ear temperature analysis of the IR images was carried out using FLIR Quick Report 1.2 SP1 253

(FLIR Systems, Oregon, USA). Exploratory data analysis, including ANOVA, REML, time 254

series analysis and regression analysis were performed using Genstat version 13.1 (VSN 255

International Ltd., UK). Bonferroni correction was applied to ANOVA post-hoc tests in pair-256

wise comparisons. Separate pots within growth cabinets were considered independent 257

replicates. A comparison of hot and cool treatments was not carried out due to insufficient 258

replication of this factor. Effects were considered significant at P<0.05. 259

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Results 260

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Ear temperature 262

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Water availability did not have a significant effect on ear temperature depression (ETD) of 264

the wheat genotypes utilised in this study (P=0.075, Figure 1). In the ‘Cool’ environment, the 265

difference in mean ETD between SB020 and SB087 was statistically significant (P=0.029), 266

whereas no significant difference was identified between SB155 and SB165 (P=0.083, Figure 267

2). In the ‘Hot’ environment there was no statistically significant difference between 268

SB020/SB087 (P=0.112) whereas SB155/SB165 showed significant differences in the mean 269

ETD (P=0.015). Genotype SB118 was not included in the IR analysis because growth did not 270

advance beyond GS45. Due to a technical fault with the thermographic equipment used, 271

SB009 was recorded incorrectly and this genotype was also excluded from IR analysis. 272

273

SB020 and SB087 only were selected for detailed ear temperature analysis on the basis of 274

Pinto et al. (2010) having identified SB020 as the higher yielding genotype of the pair under 275

conditions of heat stress, drought and irrigation (Supplementary Fig. S4). The ETD of 276

genotype SB020 decreased by 2.46°C i.e. the spike got warmer, between the period that the 277

plants were placed in the growth cabinets until the end of anthesis, with a concurrent decline 278

in florets at FDS F3 of 42%. Over that same period, the highest ETD was observed when 279

florets at FDS HF1 and HF2 were at a maximum. At FDS F3, there was no clear correlation 280

between the proportion of florets at this stage and a higher ETD (data not shown). However, a 281

decrease in ETD was observed to coincide with increasing number of florets at FDS F3 and 282

PF4 both in SB020 (P=0.012) and SB087 (P=0.032, Figure 3). There was no difference in the 283

slope of the linear relationship between the two genotypes in cool (P=0.090) or hot (P=0.303) 284

treatments. 285

286

Further, in genotypes SB020 and SB087, data relating to IR imaging and FDS of each ear 287

were evenly split into three sections, namely the ‘basal’, ‘middle’ and ‘apical’ sections 288

according to the spikelet distribution. For example, if an ear had 12 spikelets on both the even 289

and odd sides, spikelets 1-4 were labelled as ‘basal’, spikelets 5-8 were labelled as ‘middle’ 290

and spikelets 9-12 were labelled as ‘apical’. No other alternative standardised method 291

currently exists for dividing the ear into different sections. A linear regression was fitted for 292

each section of the ear to pooled data from both genotypes. There were no statistically 293

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significant differences between SB020 and SB087 in the relationships between FDS and ETD 294

in any of the three ear sections (P=0.124). Similarly, there was no difference in the slopes of 295

the linear fits between basal, middle and apical regions in cool (P=0.163) and hot (P=0.974) 296

treatments. 297

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Gas exchange 299

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Carbon dioxide and water vapour exchange of four replicate ears in genotypes SB009, 301

SB087, SB155 and SB165 was measured daily at three set time intervals for a total of three 302

days. There was no significant difference in the carbon dioxide uptake between the 303

genotypes, except during the midday session (P=0.019). Irrigation was not identified as 304

having a significant effect on the carbon dioxide uptake at any stage (Table 1). There was no 305

significant difference in the water vapour exchange between the genotypes or as a result of 306

varying levels of irrigation (Table 1). Genotypes SB009 and SB087 were utilised to study the 307

interaction between the percentage of florets at FDS F3 and the rate of gas exchange of wheat 308

ears. No significant differences were identified in either the carbon dioxide uptake or the 309

water vapour exchange between the genotypes in both the ‘Cool’ and the ‘Hot’ environments. 310

The results from genotype SB087 indicated a trend correlation between CO2 uptake and 311

florets at F3 in the ‘Cool’ environment, but this was not statistically significant (P=0.054). 312

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Discussion 328

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Early methods of breeding for crop yield improvement were based on indicators of crop 330

performance, such as ear density, fertility and grain size. These highly integrative agronomic 331

traits while being plastic in their response to environment, do not offer any information on 332

factors affecting their expression in season. However, in recent decades a number of 333

physiological processes in wheat have been linked to yield, including osmotic adjustment 334

(Blum 1988; Morgan and Condon 1986), maintaining root development to maximise soil 335

moisture extraction (Lopes and Reynolds 2010) and delaying leaf senescence (Hsiao et al. 336

1984). Under irrigated conditions Fischer et al. (1998) found that grain yields associated well 337

with canopy temperature depression (CTD), leaf conductance (LC) and leaf photosynthetic 338

rate (LPR) in genotypes developed over a 26 year period in Mexico. Canopy temperature has 339

been identified as being indicative of heat tolerance (Reynolds et al. 1998), drought tolerance 340

(Blum et al. 1989) and plant water status (Blum et al. 1982). As with most physiological 341

processes in plants, the genetic basis of CTD is likely to be complex and involve a large 342

number of interacting genes. Therefore, selecting genotypes based on genetic screening is a 343

fraught and costly approach. Reynolds (2002) concluded that screening based on CTD allows 344

for early removal of genetically inferior genotypes, which increases the accuracy and the 345

speed of the breeding process. 346

347

The lack of detailed studies identifying the exact mechanisms controlling CTD means that 348

there is an equally great gap in our understanding of the mechanisms regulating TD in the 349

ear. Teare et al. (1972) postulate that higher grain yields observed in long awned cultivars of 350

wheat, compared to short awned cultivars, can in part be explained by differences in stomatal 351

density. This is closely linked to the gaseous exchange capacity of the glumes which, due to 352

the presence of stomata, have the potential to cool the ear during sensitive periods. As glumes 353

transpire at a rate similar to that of the flag leaf, there is a possibility that the mechanism of 354

TD in the ear is regulated in a similar manner to the mechanisms controlling TD in leaves 355

(Blum 1985). This leads to a suggestion that to protect heat sensitive processes such as 356

gametogenesis and fertilisation, heat tolerant populations are capable of maintaining lower 357

ear temperatures in stressed conditions than susceptible populations, similar to the plant 358

cooling the canopy to protect sensitive developmental stages (Bahar et al. 2008). Yield data 359

were not collected from plants utilised in this pilot study as most ears were damaged to a 360

certain extent during floret scoring. Pinto et al. (2010) utilised the same genotypes in trials 361

12

with combinations of water availability and heat stress. Yield data, and particularly the 362

sensitivity of wheat genotypes to the drought and heat stress informed the choice of 363

contrasting SB genotypes in this study (Supplementary Figure S4 & Table S1). Utilising such 364

a selection of genotypes, this study shows that wheat may be capable of thermoregulation in 365

the ear, and the rate of which may be modified by flowering stage. Nevo et al. (1992) 366

concluded that in a number of genotypes of the wild progenitors of wheat (Triticum 367

dicoccoides) and barley (Horedeum spontaneum), intense thermogenesis in the flowering 368

organs occurs when a plant is exposed to temperatures outside of its optimal growing 369

conditions. This is an active attempt by the plant to adjust and adapt in order to prevent 370

damage to reproductive processes. If plants retain the capability to actively produce heat 371

though mechanisms inherent to animals in order to shield their reproductive organs from 372

stress, it is equally feasible that plants utilise a cooling mechanism to shield the flowers from 373

high temperature (McDaniel 1982). In this study, the extent of thermoregulation varied 374

among the genotypes in the ‘hot’ treatment, with SB165 expressing a significantly lower 375

AET than the other genotypes. In the case of SB165, the AET was approximately 0.6°C 376

cooler than the mean AET of the other three genotypes. Figure 2 illustrates the differences in 377

AET between the genotypes in the ‘cool’ and ‘hot’ treatments. In this context, a better 378

understanding of the interactions which explain differences between genotypes might be 379

gained by including the root network (Hurd 1968), as well as pollen production and viability 380

(Bita et al. 2011) in the consideration. This study did not attempt to correlate ear temperature 381

with the corresponding canopy/flag leaf temperature, but solely attempted to establish 382

whether TD occurred between the six paired genotypes of Seri/Babax in a controlled 383

environment setting. However, correlating ear temperature and flag leaf temperature may be 384

a key indicator as it would shed light on preferential cooling of these organs at different 385

stages of development. 386

387

The findings of this study indicate that TD does occur during the early, but not in the late 388

stages of anthesis. In the examined genotypes, differences between AET and air temperature 389

were limited to between 1.5°C and 2.0°C. In heat stressed environments, this cooling of the 390

ear has the potential to help a plant maintain key processes which would otherwise be 391

disrupted and cause irreversible damage to the yield potential. A candidate mechanism 392

identified in rice is the dehiscence of the thecae leading to pollen release; this process is 393

particularly sensitive to heat stress (Matsui et al. 2000). In maize the equivalent process of 394

pollen release is the dehydration of the stomium which releases pollen (Keijzer 1996). Hence 395

13

the wheat may have the potential to maintain a cooler ear up to the point of pollen release. 396

With a 2°C to 4°C rise in average global temperatures predicted to occur as a result of climate 397

change by the end of this century (IPCC 2007), the cooling capacity of the wheat ear may 398

have the potential to maintain ear metabolic processes despite increasing temperature. 399

400

Water availability for transpiration from the glumes may be greater in the lower sections of 401

the ear because of their proximity to the xylem transport system contained within the stem. 402

The transportation stream consists of columns of internal water created by the losses of water 403

from above ground biomass. Maintaining this stream of water to the ear as a result of 404

increased transpiration from the glumes should create a corresponding cooling effect. In this 405

study, however, there was no difference in the temperature of the ear sections due to the 406

proximity to the stem. The observed temporal increase of ear surface temperature was solely 407

due to mean FDS of each section. 408

409

Several key factors interact with heat and drought stress and modify plant response and 410

eventual yield loss e.g. concentration of WSC in plant tissue, chlorophyll content and pollen 411

development. WSC can maintain plant function and grain yields. Waite and Boyd (1953) 412

identified significant differences in the concentrations of individual WSC between plant 413

organs depending on their growth stage. Drought stress tolerant populations of wheat are 414

likely to have higher concentrations of WSC (Xue et al. 2008), increasing the potential of 415

homeostasis being maintained in the growing ear. 416

417

Chlorophyll concentration varies between genotypes and its susceptibility to heat stress 418

differs accordingly (Graham and McDonald 2001). To date, the vast majority of studies 419

dealing with genotype and chlorophyll interactions have focused largely on chlorophyll 420

concentrations in the flag leaf, not the ear. Chlorophyll is closely related with photosynthetic 421

activity, which in turn has been linked to the ability of a plant to regulate CTD. 422

423

Finally, across a wide range of crops the critical threshold for pollen production and viability 424

varies only by 2°C to 4°C between semi-arid crops (ground-nut (Rasad et al. 1999)), 425

vegetable crops (tomato (Zinn et al. 2010)) and crops grown in a flooded environments (rice 426

(Nakagawa et al. 2002)). It appears that although pollen structure differs greatly between 427

crops (Edlund et al. 2004), a crops ability to tolerate heat and drought stress does not solely 428

lie in an ability to produce pollen capable of withstanding adverse environmental conditions. 429

14

It is likely that an ability to shield pollen from these adverse conditions is the vital feature 430

that allows plants to grow in environments where the critical threshold for pollen production 431

and viability in the early stages of development is reached on a regular basis. 432

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15

Conclusion 464

465

This study has highlighted a number of key issues, namely that an active cooling mechanism 466

might have evolved in the ear to protect the heat sensitive stages of flower development and 467

that water availability. The results illustrates that TD does occur in the ear, with the potential 468

of significantly reducing the AET in stressed environments. No evidence was found to 469

support the hypothesis that the greatest TD would coincide with FDS F3, rather that it is FDS 470

HF1 and HF2 which show the greatest TD. Future work should focus on verifying the extent 471

to which the results are applicable to studies in the field: (i) do differences exist between base 472

cellular temperatures which significantly influence plant tolerance to stress; and (ii) at what 473

stage during anthesis TD in the ear is most critical. The results provide a strong platform 474

from which further work can be conducted. A much wider range of genetic material needs to 475

be fully screened in order to identify whether TD takes place in all genotypes, and to what 476

extent it correlates to stress tolerance as well as how alternative mechanisms may be used to 477

perform thermoregulation in the ear. 478

479

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16

Acknowledgements 498

499

We would like to thank Mr. Laurence Hansen, Mrs. Caroline Hadley, Mr. Benn Taylor for 500

their technical support during this study. This work was funded by the UROP scheme of the 501

University of Reading. 502

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Figure 1. Ear Temperature Depression (ETD) of wheat genotypes SB020, SB087, SB155 and 707

SB165 grown in irrigated and drought conditions. ETD was defined as the difference between 708

ambient and mean ear temperatures. Positive ETD denotes cooling of the ear relative to 709

ambient air. Bars indicate standard error. 710

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729

Figure 2. Ear Temperature Depression (ETD) of wheat genotypes SB020, SB087, SB155 and 730

SB165 grown in cool (22/12ºC) and hot (28/14ºC) environments. ETD was defined as the 731

difference between ambient and mean ear temperatures. Positive ETD denotes cooling of the 732

ear relative to ambient air. Bars indicate standard error. 733

734

735

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737

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739

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25

Figure 3. The relationship between mean Flower Development Score (FDS) of male flower 747

parts (anthers) and Ear Temperature Depression (ETD) in genotypes SB020 and SB087 in 748

cool (22/12ºC) and hot (28/14ºC) environments.. The decreasing trend of ETD is significant 749

both in the ‘hot’ (dashed lines, P=0.003) and in the ‘cool’ (solid lines, P<0.001) 750

environments. 751

752

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26

Figure 4. The relationship between mean Flower Development Score (FDS) of male flower 768

parts (anthers) and Ear Temperature Depression (ETD) in apical, middle and basal ear 769

sections. Data for genotypes SB020 and SB087 were pooled, no statistically significant 770

differences between the ear sections were found in either the ‘hot’ (panel A, P=0.975) or 771

‘cool’ (panel B, P=0.163) environments. 772

773

774

775

27

Table 1 – P-values from a REML analysis of carbon dioxide uptake (V.CO2) and water 776

vapour exchange (∆H2O) of the wheat ears in the experiment. Ear gas exchange was 777

measured for 100 sec intervals on second order ears of SB020, SB087, SB155 and SB165. 778

779

Carbon dioxide uptake (V.CO2) 780

P-values 781

Genotype Irrigation 782

Morning (09.00-11.00h) 0.266 0.413 783

Midday (12.00-14.00h) 0.019** 0.603 784

Afternoon (15.00-17.00h) 0.061 0.515 785

786

Water vapour exchange (∆H2O) 787

P-values 788

Genotype Irrigation 789

Morning (09.00-11.00h) 0.469 0.298 790

Midday (12.00-14.00h) 0.297 0.883 791

Afternoon (15.00-17.00h) 0.957 0.327 792

P-value significance levels: * - P<0.001, ** - P>0.01, *** - P>0.05. 793